The intracellular replicating form (RF) DNA of coliphage phi X174 has been shown to be heterogeneous with respect to nucleotide sequence. The mature viral form of this DNA, the mature and intracellular RF DNAs of several additional bacteriophages, and plasmid, animal virus, and mammalian mitochondrial DNAs will be examined for such nucleotide sequence heterogeneity. These DNAs, most of which exist as closed circular duplex DNAs or can be so rendered artificially, will be subjected to the introduction of a very limited number of strand breaks per molecule, denatured, renatured, closed with DNA ligase, and the closed circular fractions purified. The susceptibility of these to DNAs to the action of the highly single strand-specific Alteromonas nuclease and their examination by electron microscopy reveals whether lesions, caused by nucleotide sequences in one strand which do not have complementary sequences in the other strand, are present to an appreciable extent. Such lesions serve as substrate sites for the nuclease and appear as single-stranded "bush" regions in the electron microscope. Control samples are nicked and closed only without denaturation; the Alteromonas nuclease will not cleave the resulting closed duplexes unless lesions are present. These studies will have as their object the determination of whether heretofore unsuspected sequence heterogeneity is present in various small DNA genomes. In other studies, a possible explanation for the apparent increase in superhelical content of negatively supercoiled DNAS subjected to the introduction of a limited number of ultraviolet photoproducts will be tested. This explanation, if valid, would demonstrate an important difference between supercoiled and nonsupercoiled DNA at the DNA helix level. The dye-mediated renaturation of denatured closed circular DNA strongly alkaline solutions will also be further investigated, as will the anomalous behavior of phage PM2 closed circular DNA in alkaline solutions. The interaction of DNA topoisomerase I with denatured viral closed circular DNA to produce, among other species, oligomeric forms joined by a novel type of linkage will be further characterized and the nature of the linkage determined.